Immunoblotting Analysis of Adipose Tissue

WAT and BAT samples from all groups were homogenized in RIPA buffer as previously described^{39 (link)}, and 20 μg protein was resolved by SDS-PAGE and immunoblotted with antibodies against UCP1 (Alpha Diagnostic International, San Antonio, TX, #UCP11-A, 1:1000 dilution), PGC1α (Millipore, #AB3242, 1:1000 dilution), CPT1 (Alpha Diagnostic International, #CPT1M11-A, San Antonio, TX, 1:1000 dilution), Mitomix (Abcam, #MS604, 1:1000 dilution), p-CREB (Cell Signalling Technology, #9198, 1:1000 dilution), p-ACC (Cell Signalling Technology, #3661, 1:1000 dilution), p-ERK (Cell Signalling Technology, #9102, 1:1000 dilution) and 14-3-3 (Santa Cruz, #Sc629, 1:1000 dilution). Secondary antibodies, donkey anti-rabbit IgG-HRP (H + L) (Jackson ImmunoResearch, #711-035-152) and sheep anti-mouse IgG-HRP linked (GE Healthcare, #NA931, 1:5000 dilution) were used. Immunolabelled bands were quantified by densitometry using ImageJ software (1.38×, National Institutes of Health, USA).